HPLC Analytical Method Deveopment and Validation

Good HPLC methods must satisfy both technical requirements (sensitivity, specificity, linearity, accuracy and precision) as well as business needs (reliability in routine use and a run time appropriate to the number of samples to be tested).  These requirements are equally important in both a development and routine QC context: decisions during drug development must be based on reliable data, and routine QC testing, including stability studies, must control risks to product quality and patient safety. 
 
This course presents a logical, step-wise approach to the development of HPLC methods (Day 1) and then explains how to validate chromatographic methods in line with regulatory expectations and best practice (Day 2). 
 
The course is intended for analytical scientists who have experience of operating HPLC instrumentation.
Course Programme
Day One
Revision of chromatographic theory

• Separation modes
• Factors affecting resolution
• Peak symmetry
• Band broadening
• The effect of particle size and extra-column volume on efficiency

Important chemical concepts

• Factors affecting analyte/stationary phase interactions (polarity, hydrogen bonding and pKa)
• Stationary phase endcapping

Analyte properties affecting pKa, solubility and detectability
Workshop: reading solute structures
Matrix properties

• Effect on extraction
• Chemical interference
• Selectivity and detector wavelength
• Injection solvent strength

Method performance requirements

• Defining method performance requirements
• Measurement uncertainty vs. specification limits

Sample preparation

• Selective sample preparation
• Choice of filter membrane
• Chemical and physical stability of samples

Developing the separation

• Starting conditions for different separation types
• *  Neutral/ionisable organic molecules
• *  Special cases
• Separation modes: isocratic, gradient, ion pairing/suppression, HILIC, aqueous normal phase, normal phase, ion exchange and size exclusion
• Retention mechanisms
• Choice of stationary phase (including silanol activity considerations)
• Mobile phase pH and solute pKa
• Choice of pH buffer
• Temperature effects
• Core-shell and UHPLC columns
• Detector selection

Gradient elution

• When to use gradient elution
• Significance of gradient delay volume
• Retention and resolution models in gradient separations
• Gradient profile optimisation
• The effect of column dimensions and temperature
• Step-wise gradient method development strategy

Case studies
Workshop: selecting starting conditions for method development
 
Day 2 – HPLC Method Validation
Regulatory guidance (ICH, US, EU, WHO)
Validation terminology
Setting meaningful acceptance criteria

• Acceptance criteria based on specification limits
• Measurement uncertainty and sources of error
• Typical acceptance criteria
• Lifecycle approach to method validation: Analytical Target Profile (ATP) and analytical control strategy

Experimental approaches to method validation

• Specificity: with and without impurity standards
• Linearity: best practice
• Use of spiking experiments
• Options for evaluating sensitivity
• Recommended robustness experiments

Phase-appropriate method validation
 
Workshop – setting method validation acceptance criteria
Writing effective analytical methods, validation protocols and reports

• Pre-validation check-list
• Contents of method and validation documentation
• Mistake-proofing analytical methods

Dealing with validation failures
Setting system suitability criteria

• Regulatory guidance
• Statistically-based methods

Verifying compendial procedures

• Regulatory guidance
• Approaches for different method types

Workshop – planning a method validation exercise            
                       
 
 

DoubleTree Hilton Hotel Angel and Kings Cross

DoubleTree Hilton Hotel Angel and Kings Cross, 60 Pentonville Road, London, N1 9LA, United Kingdom